Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. During the previous funding cycle, work generated an innovative new structural model for TPH, provided insights into how the hydroxylases assemble into tetramers, identified a new phosphorylation site on TPH, and provided important information in the relative stabilities of TH and TPH. Experiments planned for the next funding period will address the need for more detailed information with a particular focus on four key areas. (1) Using the new structural model of TPH, along with available crystal coordinates for TH, sequence elements will be mapped within the active sites of the enzymes. These experiments will identify amino acid residues that influence and direct differential substrate utilization. (2) Mutagenesis will be conducted to refine the determinants of tetramer formation and the role of macromolecular assembly in regulating enzyme activity. Notably, while monomers have been demonstrated to retain activity, it remains to be determined how tetramer formation might influence the post-translational regulation of enzyme activity. (3) The hypothesis will be tested that differences in N-terminal regulatory domain sequences are responsible for enzyme stability. (4) The novel hypothesis will be explored that phosphorylation-regulation of TPH involves issues of selective stability as well as targeted interactions with the chaperone protein, 14-3-3. The proposed experiments extend the previous successes in the characterization of TH and TPH structure-function analysis. The studies will develop several novel ideas concerning the regulation of these enzymes and increase the base of knowledge from which we can interpret new information that will emanate from the human genome initiative in terms of naturally occurring polymorphisms in these important enzymes.